Biology Reference
In-Depth Information
Fractionation is achieved by manipulation of
(a) the mobile phase properties such as slope
(time) of the gradient, organic modi
TABLE 1
Proteome analysis: intact proteins or protein
digests (peptides)?
er concen-
tration, buffer pH, salt concentration, and/or
ampholyte pH range; (b) type of column:
reversed-phase (e.g., C-18, C-8), ion exchange,
size exclusion, normal phase (e.g., silica), or
af
Protein mixtures are
complex
Peptide mixtures are more
complex
Proteins are less soluble
Peptides are more soluble
Proteins are harder to
handle
Peptides are easier to work
with
nity (use of antibody, lectin, aptamers, metal,
DNA, etc.); and (c) gel properties. Therefore,
experiments are usually designed to separate
proteins and metabolites based on their size,
hydrophobicity, polarity,
Loss of one protein
is loss of information
Loss of one peptide is not
loss of a protein or
information
charge,
isoelectric
Protein analysis by MS
requires sophisticated
instruments
Peptide analysis is done
with simpler MS
instrumentation that is more
accessible
point, or af
nity.
Chromatography, especially HPLC in its
different modes of separation, has been used
extensively for the fractionation of complex
mixtures of proteins, peptides, and metabolites.
An advantage of HPLC is that it allows
the selective fractionation of a mixture by
manipulation of the mobile and/or stationary
phases. The principle of chromatographic frac-
tionation is based on the interaction of the
compounds of interest with the solid support
(stationary phase) and the mobile phase. The
interaction may be adsorption on silica surfaces,
partitioning on reversed phase materials, or ion
exchange based on effective charge of the
proteins, peptides, and metabolites. Fraction-
ation is achieved by using mobile phase gradi-
ents, whereby the compounds that are
introduced into the head of the column are
differentially eluted by changing the organic
modi
Protein and metabolite mixtures are fraction-
ated based on their physical and chemical prop-
erties by a variety of chromatographic and
electrophoretic methods (
Table 2
).
TABLE 2
Various proteomic and metabolomic
fractionation procedures based on
chemical/physical properties
Fractionation Method
Property
Ultracentrifugation
Density
Size exclusion
chromatography (SEX)
Stoke
'
s radius
Isoelectric focusing (IEF)
Isoelectric point
er concentration with time (RP and
HILIC chromatography), the salt content with
time (hydrophobic interaction chromatography
and ion exchange [IEX]), or by mobile phase
pH gradient (IEX chromatography). The frac-
tionation of peptides has been mostly con-
ducted using SCX, a procedure that was
popularized by John Yates and his coworkers
2
when
Hydrophobic interaction
chromatography (HIC)
Hydrophobicity
Hydrophilic interaction
chromatography (HILIC)
Polarity
Reversed-phase
chromatography (RP)
Hydrophobicity
Ion exchange
chromatography (IEX)
Charge
they
introduced multidimensional
protein identi
cation technology (MudPIT).
Af
nity chromatography
Speci
c biomolecular
interaction
The acidi
ed peptide mixture is loaded onto
a biphasic SCX/RP column. Discrete peptide
fractions are displaced from the SCX column
Gel electrophoresis (GE)
Stoke
'
s radius
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